A new global pandemic disease named COVID–19 has emerged and is still spreading at alarming rates at the time of this report. COVID–19 can cause severe symptoms such as damaging inflammatory response, fever or severe respiratory illness and lead to death. The causative agent of COVID–19 was found to be a novel coronavirus closely related to the severe acute respiratory syndrome coronavirus (SARS-CoV) based on the latest phylogenetic analysis1–3. There are nonetheless some major essential differences in their genetic make-up that led to their very different behaviors. Indeed, SARS-CoV–2, as it is called now, appears to have a high transmissibility from person to person and antibodies that could inhibit SARS-CoV are not functional on SARS-CoV–22,4–5. Despite global efforts, we still lack an effective antiviral strategy, drug or vaccine to fight off this virus with the growing fear that SARS-COV–2 may become another endemic virus in our communities.
In order to lower the costs of and speed up the drug discovery phase, numerous researchers have utilized in silico tools such as protein-ligand docking software to screen for traditional compounds that could bind to and inhibit the key proteins present in SARS-CoV–2, highlighting their potential antiviral activity6. The major targets for these compounds include SARS-CoV–2 key proteins 3-chymotrypsin-like protease (Mpro), papain like protease (PLpro), RNA-dependent RNA polymerase (RdRp), small envelope protein (E), membrane protein (M) and spike (S) proteins. The S proteins interact directly with human angiotensin-converting enzyme (ACE2), allowing the virus to enter the cells. The S protein is a class I fusion protein consisting of S1 and S2 domains with the receptor binding domain (RBD) located on the S1 domain4. The RBD is the main target of antibodies and fusion inhibitors in development such as the human convalescent COVID–19 patient-origin B38 antibody (B38) and plant lectin griffithsin (GRFT).
Here, we report the in silico potent antiviral activity against SARS-CoV–2 of a previously discussed novel broad spectrum anti-infective fusion protein between a mutant of the ricin A chain and pokeweed antiviral protein isolated from leaves (RTAM-PAP1) from Ricinus communis and Phytolacca americana respectively7. RTAM-PAP1 activity was compared with that of the B38, ricin A chain (RTA), pokeweed antiviral protein isolated from leaves (PAP1) and GRFT. Their binding and inhibiting capacities were evaluated against the major key proteins of SARS-CoV–2 using the latest peptide-ligand docking software8–13.
The 3D structure of RTAM-PAP1 was obtained as previously described7 and those of RTA, PAP1, B38, and GRFT were retrieved in protein data bank (PDB) format from the Research Collaboratory for Structural Bioinformatics (RCSB) website (https://www.rcsb.org/). A knowledge-based scoring docking prediction was done for all the compounds against S, S1 RBD and M using CoDockPP global docking. An additional run was done for ACE2 and human SARS-CoV antibody CR3022 against S1 RBD as reference5. The 3D structures of all the key proteins and ACE2 were already available at the software site in this “covid–19 targets docking only” version. The peptide/antibody-ligand version was used as small molecules docking software is not suited for these types of compounds. The generated 3D model of B38, ACE2 and CR3022 bound to S1 RBD were comparable to available crystallography of the same complexes in RCSB (access: 7BZ5, 6M0J and 6W41 respectively) with some deviations (results not shown). However, the greater binding affinity and fusion inhibiting activity of B38 compared to CR3022 for S1 RBD was observed in accordance with published in vitro results1,3,5. Indeed, B38 was found to have a dissociation constant of 70.1 nM with a complete inhibition of ACE2 binding to S1 RBD compared to CR3022’s dissociation constant of 115 nM with no inhibition of ACE2 binding. The difference in inhibition of ACE2 binding to S1 RBD is due to their binding conformation to S1 RBD. However, ACE2 binding to S1 RBD was found to have the smallest dissociation constant in literature with a value ranging from 4 to 15 nM. The results for the first and last model (out of the top ten generated) of each compounds in complex with S, S1 RBD and M are presented in Table 1.a. B38 has the highest overall binding affinity of the lot with a binding energy ranging from –449 to –300 kcal/mol, as expected. ACE2’s binding energy was between –314 to –246 kcal/mol for S1 RBD. RTAM-PAP1 is very comparable to B38, with an overall higher binding affinity (lower binding energy) than all of the other compounds tested against the S, S1 RBD and M key proteins, sometimes higher than that of B38 with –469 kcal/mol for M, for example. The high binding affinity of RTAM-PAP1 and B38 to S, S1 and M may be explained by the fact that the M epitope is very similar in structure to S1 RBD (figure 1.a)1,3. RTA binding affinity is similar to RTAM-PAP1 to a certain extent and GRFT and PAP1 are very comparable. The same higher binding affinity behavior for RTAM-PAP1 was observed with Mpro, PLpro, E and RdRp when compared to PAP1, GRFT and RTA (Table 1.b). All of the tested compounds showed potentially inhibiting biding conformations to the various key proteins based on the 3D structures of the complexes formed (results not shown). These results indicate that the fusion between RTAM and PAP1 allowed RTAM-PAP1 to be more stable across the different possible binding conformations with a higher binding affinity than either of its moieties alone when in complex with SARS-CoV–2 key proteins.
B38 was found to have an inhibition of the cytopathic effect of 50% (EC50) against SARS-CoV–2 simultaneous infection in Vero Cells in vitro at the concentration of 0.177 ug/ml. It was further demonstrated that B38 was effective in mice post-infection1. GRFT was found to have low pre-infection EC50 on different strains of SARS-CoV in cytoprotection (CPE) assays in vitro (0.6—1.2 ug/ml) and effective in mice pre-infection14. RTA was shown in literature to have high binding affinity to many viral proteins15–16. PAP1 has a broad range antiviral activity against numerous infections both in vitro and in clinical trials17–18. An earlier different version of RTAM-PAP1 was shown to have potent broad range antiviral activity at low post-infection EC50 (0.002—12.3 ug/ml) against human immunodeficiency virus-I (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (Zika) and human coronavirus 229E (HCoV229E) in CPE assays in vitro7,19 . RTA and PAP1 have been observed to have a drastic increase in viral inhibition activity if administered pre-infection both in vitro and in vivo at sub-toxic dosages20–23 with potent antiviral mechanisms, from viral DNA/RNA depurination, viral proteins synthesis inhibition, viral cell entry inhibition to apoptosis induction of infected cells via a preferential virus infected cell entry mechanism7.
Nonetheless, such high affinity of RTAM-PAP1 to many key proteins of SARS-CoV–2 is uncommon. Yet, the most surprising part of the generated models was the discovery of unique binding mechanisms of RTAM-PAP1 with potential inhibiting activity by hindering viral entry and cellular machinery. This discovery might explain the previously observed gain of function of RTAM-PAP17 by means of the acquired ability to simultaneously bind the target with both moieties with high affinity, i.e. increasing the docking sites from 86 to 102 for single moiety binding and simultaneous binding to S1 RBD respectively, for example.
In order to confirm these findings, RTAM-PAP1 was run against SARS-CoV–2 S1 and M using different docking programs (Zdock and HADDOCK2.2) with the known active residues in RCSB. The synergetic binding of RTAM-PAP1 was confirmed and the generated models for M are shown in figure 1.b-d. Although the model generated by HADDOCK2.2 returned a more important role for PAP1 than RTAM, the simultaneous binding of both moieties can clearly be seen when in complex with M with an increase in docking sites from 62 for single moiety binding to 96 for simultaneous binding of both moieties (Zdock model). This might significantly increase RTAM-PAP1 anti-SARS-CoV–2 activity. It can be concluded from these results and those previously acquired in vitro that the fusion of RTAM and PAP1 via the flexible linker conferred greater structure stability, enhanced activities, new binding sites and mechanisms and also, potentially, novel functions to RTAM-PAP1.
For those reasons, a short toxicity study of RTAM-PAP1 was conducted in BALB/c mice to determine the potential maximum tolerated dose. Adverse clinical signs were observed at a single bolus intravenous administration of 3 mg/kg of RTAM-PAP1 with up-regulation of IP–10, KC and MCP–10 chemokines from 14 cytokines/chemokines assessed (results not shown). These results are in line with previously described homopolymers of ribosome inactivating proteins and confirm an in vivo behavior intermediate between that of native ribosome inactivating proteins and of immunotoxins24–25.
In conclusion, given the very high affinity for SARS-CoV–2 key proteins, the previous antiviral results in vitro, the newly discovered mechanisms, the preliminary in vivo profile, potent bioactivities across the assays and preferential entry into virus infected cells as opposed to non-infected cells, it is the opinion of the authors that this novel N-glycosidase ribosome inactivating fusion protein be tested against SARS-CoV–2 in vitro and in vivo. It would be the first tested therapeutic utilizing this particular strategy against COVID–19 and might make a difference at subtoxic dosages and open the doors for the discovery of novel SARS-CoV–2 targets and therapeutic protein structures.